Thyrotropin-releasing hormone receptor 2(TRHR-2)

ABSTRACT

The present invention is directed to the novel receptor for TRH which has been designated as TRH receptor 2. The invention encompasses both the receptor protein as well as nucleic acids encoding the protein. In addition, the present invention is directed to methods and compositions which rely upon either TRHR-2 proteins or nucleic acids.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application represents U.S. National stage of internationalapplication PCT/SE97/01999, which has an international filing date ofNov. 28, 1997, and which was published in English under Article 21(2) ofthe PCT on Jun. 11, 1998; The international application claims priorityto Swedish Application 9604439-1, which was filed on Dec. 2, 1996.

FIELD OF THE INVENTION

The present invention is in the field of biological receptors and thevarious uses that can be made of such receptors. More specifically, itrelates to nucleic acids encoding a novel receptor forthyrotropin-releasing hormone and to the receptor protein itself.

BACKGROUND OF THE INVENTION

Thyrotropin-releasing hormone (TRH) is a tripeptide (pyroglutamicacid-histidine-proline-amide) present in the central nervous system(thalamus, cerebral cortex, spinal cord) as well as in the periphery(pancreas, gastrointestinal tract, placenta). In the hypothalamus, TRHis synthesized by peptidergic neurons of supraoptic and paraventricularnuclei. It is then axonally transported to the median eminence where itis stored. Upon secretion into the bloodstream, TRH is transported tothe pituitary where it stimulates the production of thyroid stimulatinghormone (TSH) which, in turn, stimulates the production of thyroxin (T4)in the thyroid gland (Gaillard, in Pharmacologie: Des ConceptsFondamentaux Aux Applications Thérapeutiques, M. Shorderet ed., pp.415-448 (1992)).

In addition to its role in regulating the synthesis and secretion ofhormones from the anterior pituitary, there is evidence that TRH acts asa neurotransmitter (Wu, et al., Neurosci. Let. 142:143-146 (1992)). TRHis found abundantly in the central nervous system and exogenousadministration of TRH elicits a variety of behavioral changes. Itproduces a rapid onset, neurotransmitter-like, excitation of spinallower motor neurons and reduces neurological deficits observed aftertraumatic spinal cord injury in cats.

The distribution of TRH-containing cells, fibers or receptors suggests apotential role for TRH in the perception of noxious stimuli.Specifically, TRH is present in the periaqueducal gray (PAG), the nucleiraphe magnus (NMR), in the pallidus and dorsal horn of the spinal cord.TRH binding sites have been found in the brain, pituitary, dorsal andventral horns of the spinal cord, and in peripheral tissues. Wheninjected centrally (I.C.V. and I.C.), TRH induces a short lastingsupraspinal antinociception. The analgesia induced by I.C.V. TRHinjection is twice as great, on a molar basis, as that induced bymorphine (Boschi, et al., Br. J. Pharmacol. 79:85-92 (1983)). Thisantinociceptive effect is detected in models of chemically andmechanically, but not thermally, induced pain.

The actions of TRH are mediated by the stimulation of specific cellsurface receptors. There is evidence that TRH receptors found in thepituitary transmit their signal to the cell interior through a G proteinto trigger the inositol phospholipid-calcium-protein kinase Ctransduction pathway (Straub, et al., Proc. Nat'l. Acad. Sci. U.S.A.87:9514-9518 (1990); Duthie, et al., Mol. Cell. Endocrinol. 95:R11-R15(1993)). A cDNA sequence encoding a G protein-coupled TRH receptor wasfirst isolated from mouse pituitary cells using an expression cloningstrategy (Straub, et al., Proc. Nat'l. Acad. Sci. U.S.A. 87:9514-9518(1990)). Subsequently, several groups have described the cloning of ratTRH receptor cDNAs expressed in either a pituitary tumor cell line (GH3)or in pituitary gland (Duthie, et al., Mol. Cell. Endocrinol. 95:R11-R15(1993); De La Pena, et al., J. Biol. Chem. 267:25703-25708 (1992)). Inaddition, two isoforms of the rat TRH receptor have been shown to begenerated from a single gene by alternative splicing (De La Pena, etal., J. Biol. Chem. 267:25703-25708 (1992)).

In addition to receptors isolated from the mouse and rat, a human TRHreceptor cDNA has been cloned by Matre et al. (Biochem. Biophys. Res.Comm. 195:179-185 (1993)). With the exception of its C-terminal region,the predicted amino acid sequence of the human receptor was found to bemore than 95% homologous to its counterparts from the rat and mouse.

Using synthetic TRH analogues, a dissociation of endocrine and CNSeffects has been observed, suggesting that subtypes of TRH receptor mayexist. Certain analogues were found to affect sleeping time andbreathing frequency in test animals even though they failed to bind topituitary or brain receptors and had no measurable TSH release activity(Alexandrova, et al., Gen. Physiol. Biophys. 10:287-297 (1991)). Otheranalogues, modified in the C-terminal region, have been identified whichare ineffective in treating traumatic spinal cord injury but whichmaintain the same endocrine effects as normal TRH (Faden, Brain Research486:228-235 (1989)).

The existence of distinct TRH receptor subtypes has also been suggestedby biochemical experiments. Specifically, TRH receptors isolated fromthe brain were found to have an isoelectric point of 5.5 whereas thoseisolated from the pituitary were found to have an isoelectric point ofonly 4.9. One possible explanation for this difference is that thereceptors in the brain and those in the pituitary have different aminoacid sequences (Burt, Ann. NY Acad. Sci. 553:188 (1989)). In addition,electrophysiological experiments and measurements of intracellularcalcium concentration have suggested that TRH and TRH metabolitespresent in the brain may act by binding to different subtypes of TRHbinding sites (Toledo-Aral, et al., J. Physiol. 472:327-340 (1993)).

Therapeutically, it is clear that agonists and antagonists of TRHbinding have potential value in regulating endocrine function,controlling pain, and in the treatment of spinal cord injury. Theability to identify such agents will depend upon the availability ofpurified TRH receptors suitable for binding assays. Such assays could beused to screen for TRH agonists and antagonists; to determine the extentto which a patient's plasma contains an appropriate level of bindingactivity; and to help monitor the purity and effectiveness of agents atall stages of drug development.

SUMMARY OF THE INVENTION

To date, the only TRH receptor which has been cloned has been designatedTRHR-1. The present invention is based upon the discovery of a newreceptor for TRH which differs from TRHR-1 in terms of structure, tissuedistribution and binding characteristics. Thus, in its first aspect, theinvention is directed to a protein, except as existing in nature,comprising the amino acid sequence consisting functionally of thesequence of SEQ ID NO:2. The term “consisting functionally of” refers toproteins in which the sequence of SEQ ID NO:2 has undergone additions,deletions or substitutions which do not substantially alter thefunctional characteristics of the receptor. The term is intended toencompass proteins having exactly the same amino acid sequence as thatof SEQ ID NO:2, as well as proteins with sequence differences that arenot substantial as evidenced by their retaining the basic, qualitativeligand binding properties of TRHR-2.

The invention also encompasses substantially pure proteins withsequences consisting essentially of that of SEQ ID NO:2; antibodies thatbind preferentially to such proteins (i.e., antibodies having at least a100-fold greater affinity for TRHR-2 than any other protein); andantibodies made by a process involving the injection of apharmaceutically acceptable preparation of TRHR-2 into an animal capableof antibody production. In a preferred embodiment, monoclonal antibodyto TRHR-2 is produced by injecting the pharmaceutically acceptablepreparation of TRHR-2 into a mouse and then fusing mouse spleen cellswith myeloma cells.

The invention is also directed to a substantially pure polynucleotideencoding a protein comprising the amino acid sequence consistingfunctionally of SEQ ID NO:2. This aspect of the invention encompassespolynucleotides encoding proteins consisting essentially of the aminoacid sequence of SEQ ID NO:2, expression vectors comprising suchpolynucleotides, and host cells transformed with such vectors. Alsoincluded is the recombinant TRHR-2 protein produced by host cells madein this manner. Preferably, the polynucleotide encoding TRHR-2 has thenucleotide sequence shown in SEQ ID NO:1, and the vectors and host cellsused for expression of TRHR-2 also use this particular polynucleotide.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to bind to TRHR-2. This assayis performed by incubating a source of TRHR-2 with a ligand known tobind to the receptor and with the test compound. The source of TRHR-2should be substantially free of other types of TRH receptors such asTRHR-1, e.g., greater than 90% of the TRH receptors present in thesource should correspond to TRHR-2. Upon completion of incubation, theability of the test compound to bind to TRHR-2 is determined by theextent to which ligand binding has been displaced. The preferred ligandis either TRH or TRH which has been labelled with a detectable compound.The preferred source of TRHR-2 for use in the assay is a celltransformed with a vector for expressing the receptor and comprising apolynucleotide encoding a protein consisting essentially of the aminoacid sequence of SEQ ID NO:2. Instead of using cells in the assay, amembrane preparation can be prepared from the cells and this can be usedas a source of TRHR-2. Although not essential, the assay can beaccompanied by a determination of changes in a second messenger, e.g.changes in the intracellular concentration of calcium. This should helpto determine whether a test compound or analogue that binds to TRHR-2 isacting as an agonist or antagonist of TRH.

In another aspect, the present invention is directed to a method forassaying a test compound for its ability to alter the expression of theTRHR-2 gene. This method is performed by growing cells expressingTRHR-2, but substantially free of other TRH receptors, in the presenceof the test compound. Cells are then collected and the expression ofTRHR-2 is compared with expression of control cells grown underessentially identical conditions but in the absence of the testcompound. In a preferred embodiment, the cells expressing TRHR-2 arecells transformed with an expression vector comprising a polynucleotidesequence encoding a protein consisting essentially of the amino acidsequence of SEQ ID NO:2. A preferred test compound is an oligonucleotideat least 15 nucleotides in length and comprising a sequencecomplimentary to a sequence shown in SEQ ID NO:1. The preferred methodfor determining receptor expression is by means of a receptor bindingassay.

Definitions

The description that follows uses a number of terms that refer torecombinant DNA technology. In order to provide a clear and consistentunderstanding of the specification and claims, including the scope to begiven such terms, the following definitions are provided.

Cloning vector: A plasmid or phage DNA or other DNA sequence which isable to replicate autonomously in a host cell, and which ischaracterized by one or a small number of restriction endonucleaserecognition sites. A foreign DNA fragment may be spliced into the vectorat these sites in order to bring about the replication and cloning ofthe fragment. The vector may contain a marker suitable for use in theidentification of transformed cells. For example, markers may providetetracycline resistance or ampicillin resistance.

Expression vector: A vector similar to a cloning vector but which iscapable of inducing the expression of the DNA that has been cloned intoit, after transformation into a host. The cloned DNA is usually placedunder the control of (i.e., operably linked to) certain regulatorysequences such as promoters or enhancers. Promoter sequences may beconstitutive, inducible or repressible.

Substantially pure: As used herein, “substantially pure” means that thedesired product is essentially free from contaminating cellularcomponents. Contaminants may include, but are not limited to, proteins,carbohydrates or lipids. One method for determining the purity of aprotein or nucleic acid is by electrophoresing a preparation in a matrixsuch as polyacrylamide or agarose. Purity is evidenced by the appearanceof a single band after staining.

Host: Any prokaryotic or eukaryotic cell that is the recipient of areplicable expression vector or cloning vector is the “host” for thatvector. The term encompasses prokaryotic or eukaryotic cells that havebeen engineered to incorporate a desired gene on its chromosome or inits genome. Examples of cells that can serve as hosts are well known inthe art, as are techniques for cellular transformation (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. ColdSpring Harbor (1989)).

Promoter: A DNA sequence typically found in the 5′ region of a gene,located proximal to the start codon. Transcription is initiated at thepromoter. If the promoter is of the inducible type, then the rate oftranscription increases in response to an inducing agent.

Complementary Nucleotide Sequence: A complementary nucleotide sequence,as used herein, refers to the sequence that would arise by normal basepairing. For example, the nucleotide sequence 5′-AGAC-3′ would have thecomplementary sequence 5′-GTCT-3′.

Expression: Expression is the process by which a polypeptide is producedfrom DNA. The process involves the transcription of the gene into mRNAand the translation of this mRNA into a polypeptide.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the TRHR-2 receptor protein,genetic sequences coding for the receptor, a method for assayingcompounds for their ability to bind to TRHR-2 and a method for assayingcompounds for their ability to alter TRHR-2 expression. The receptor andthe nucleic acids encoding the receptor may be distinguished from allknown TRH receptors based upon structure, tissue distribution andbinding characteristics. With respect to structure, the relationshipbetween TRHR-2 and other TRH binders is shown in FIG. 2. The greatesthomology was observed between TRHR-2 and the human TRHR-1 receptor. Intransmembrane regions, the sequence homology between these receptorsranged from 52% to 80%. The alignment of TRHR-2 relative to other Gprotein-coupled receptors, or other members of the neuropeptide receptorsubfamily, indicates a unique sequence indicative of a newlycharacterized receptor.

It will be understood that the present invention encompasses not onlythe sequence identical to that shown in FIG. 1, but also sequences thatare essentially the same and which produce a receptor retaining thebasic binding characteristics of TRHR-2. Thus, the invention relates toproteins comprising amino acid sequences consisting functionally of thesequence of SEQ ID NO:2. In this regard, it is well known thattechniques such as site-directed mutagenesis may be used to introducevariations in a protein structure. Variations in TRHR-2 introduced bythis or some similar method are encompassed by the invention to theextent that such variant receptors retain the ability to preferentiallybind to TRH or TRH-like peptides.

The TRHR-2 receptor may also be distinguished from similar proteinsbased upon its binding characteristics. pGlu-His-Pro-Gly does not showany binding to GH4C1 cells but displays a Ki value of 640 nM to theTRHR-2 receptor. This suggests that TRHR-2 is less susceptible thanTRHR-1 to C-terminal modification of the ligand.

In addition, TRHR-2 may be distinguished from other receptors for TRHbased upon its tissue distribution. In situ hybridization studiesperformed on the rat have indicated a distinct distribution of TRHR-2 inthe pituitary gland, spinal cord, and brain. In the pituitary gland,moderate levels of TRHR-1 mRNA have been observed throughout theanterior lobe whereas both the posterior and intermediate lobes appearto be devoid of expression. In contrast, no specific hybridizationsignal was detected for TRHR-2 in the pituitary.

In the CNS, TRHR-2 mRNA is distributed throughout the entire dorsal hornof the spinal cord whereas TRHR-1 is located in sparsely distributedneurons of the ventral horn (Zabavnik, et al., Neuroscience 53:877-887(1993)). This is consistent with experiments in which autoradiographywas used to detect ³H-TRH binding sites and which suggest that TRHreceptors are expressed in both the dorsal and ventral horns of thespinal cord (Manaker, et al., J. Neurosci. 5:167-174 (1985)). In thebrain, it appears that TRHR-2 mRNA is present in much higher levels thanthe mRNA for TRHR- 1. In particular, TRHR-1 mRNA expression was observedonly at very low levels in the piriform cortex, amygdala and discreethypothalamic nuclei (superchiasmatic nucleus, SCN; ventromedialhypothalamic nucleus, VMH; paraventricular hypothalamic nucleus, PVN;and anterior hypothalamic area posterior part, AHP). In no case, withthe possible exception of the amygdala, was TRHR-1 mRNA detected inregions enriched in TRHR-2 such as the thalamus, medial habenularnucleus, frontal and parietal cortices, the pontine nucleus, or thecerebellum.

The pattern of TRHR-2 expression within the rat CNS suggests theinvolvement of at least two distinct modalities: somatosensory (possiblyincluding pain transmission) and motor. The restricted localization ofTRHR-2 mRNA throughout the entire dorsal horn of the spinal cord,reticular formation and somatosensory nuclei of the thalamus (VPL, VPM)is consistent with ascending pathways such as the spinothalamic andtrigeminothalamic tracts (pain and crude touch) as well as the mediallemniscal system (discriminative touch). The presence of high levels ofTRHR-2 restricted to the pontine nucleus and the cerebellum isconsistent with a role in motor control and/or proprioception. Thesereceptors may also be the anatomical substrate for the previouslydescribed TRH effects on motor control (see Engle, et al., The Lancet83841:73-75 (1983)). To date, only very low levels of TRH peptide or TRHbinding sites have been reported in the cerebellum suggesting that analternate ligand, as yet unidentified, may also bind to this receptor.

I. Nucleic Acids Coding for TRHR-2

As discussed above, DNA sequences coding for TRHR-2 are expressed in avariety of tissues, any of which may serve as a source for the isolationof nucleic acid coding for the receptor. The preferred source is thespinal cord of the rat, but spinal cord tissue from other species may beused as well. In addition, cells or cell lines expressing TRHR-2 mayserve as a source for nucleic acid. These may either be cultured cellsthat have not undergone transformation or cell lines specificallyengineered to express recombinant TRHR-2.

Many methods are available for isolating DNA sequences and may beadapted for the isolation of TRHR-2 nucleic acid (see for exampleSambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Press (1989)). As discussed in the Examples, a preferredmethod of isolation is to use reverse transcription PCR on mRNA isolatedfrom rat spinal cord to produce probes and to then use these probes toscreen a cDNA library. The preferred primers for carrying out the PCRamplification are:

TM3-4: 5′-AT(C or T)(A or G)(C or G)(C or T)(A or G)TIGAI(A or C)G(A orG)TA-3′ (SEQ ID NO:3)

TM7-4: 5′-(A or C)(A or T)GG(C or T)(A or G)TAGAI(C or G)AI(A or C)GG(Aor G)TT-3′ (SEQ ID NO:4)

After having produced cDNA by reverse transcription, the above primersare used for amplification. This should result in partial amplificationof the THRH2 cDNA and produce fragments suitable for screening cDNAlibraries.

Although the procedure above is known to be suitable for obtainingTRHR-2 nucleic acid, it is expected that alternative techniques can bedeveloped with relatively little effort. Thus, cDNA libraries may bescreened using probes synthesized based upon the TRHR-2 sequence shownin FIG. 1. In general, probes should be at least 14 nucleotides long andshould not be selected from regions known to be highly conserved amongproteins, e.g., the transmembrane domains of G-protein linked receptors.Alternatively, using the sequence shown in FIG. 1, it should be possibleto select different PCR primers and amplify the full length TRHR-2sequence. The same techniques that have proven successful in the rat canbe used to obtain TRHR-2 sequences from other species, e.g., from cellsor tissues derived from humans.

II. Production and Isolation of TRHR-2 Recombinant Protein

In order to express recombinant TRHR-2, a DNA encoding the structuralsequence for the protein described above must be placed in a vectorcontaining transcriptional and translational signals recognizable by anappropriate host. The cloned TRHR-2 sequences, preferably indouble-stranded form, are inserted into the expression vector inoperable linkage, i.e., they are positioned so as to be under thecontrol of the vector's regulatory sequences and in such a manner thatmRNA is produced that is translated into the TRHR-2 amino acid sequence.

Expression of the TRHR-2 receptor protein in different hosts may resultin different post-translational modifications that can, potentially,alter the properties of the receptor. Preferably, nucleic acid encodingTRHR-2 is expressed in eukaryotic cells, especially mammalian cells.These cells provide post-translational modifications which, inter alia,aid in the correct folding of the receptor protein. An appropriatevector, pCDNA3-THR2, and host, HEK293 cells, are described under“Examples.”

Other mammalian cells that may be used include, without limitation,NIH-3T3 cells, CHO cells, HeLa cells, LM(tk⁻) cells, etc. Vectorssuitable for use in each of the various cell types are well known in theart (see e.g., Sambrook, et al., supra). Preferred eukaryotic promotersinclude that of the mouse metallothionein I gene; the TK promoter ofHerpes virus; the SV40 early promoter; and the yeast GAL4 gene promoter.Some examples of suitable prokaryotic promoters include those capable ofrecognizing T4 polymerases, the P_(R) and P_(L) promoters ofbacteriophage lambda, and the trp, recA, heat shock and lacZ promotersof E coli. Expression vectors may be introduced into host cells bymethods such as calcium phosphate precipitation, microinjection orelectroporation. Cells expressing the TRHR-2 receptor can be selectedusing methods well known in the art. One simple method for confirmingthe presence of the receptor nucleic acid in cells is to perform PCRamplification using the procedures and primers discussed above. Thepresence of functional receptor may be confirmed by performing bindingassays using labelled TRH.

Once cells producing recombinant TRHR-2 receptor have been identified,they may be used in either binding assays or in assays designed toidentify agents capable of altering TRHR-2 expression. Alternatively,membranes may be isolated from the cells and these may be used inreceptor binding assays.

III. Antibodies to TRHR-2

The present invention is also directed to antibodies that bindpreferentially to TRHR-2 and to a process for producing such antibodies.Antibodies that “bind specifically to TRHR-2” are defined as those thathave at least a 100-fold greater affinity for TRHR-2 than for any otherprotein, including TRHR-1. The process for producing such antibodies mayinvolve either injecting the TRHR-2 protein itself into an appropriateanimal or, preferably, injecting short peptides made to correspond todifferent regions of TRHR-2. The peptides should be at least five aminoacids in length and should be selected from regions believed to beunique to the TRHR-2 protein. Thus, highly conserved transmembraneregions should generally be avoided in selecting peptides for thegeneration of antibodies.

Methods for making and detecting antibodies are well known to those ofskill in the art as evidenced by standard reference works such as:Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, N.Y. (1988); Klein, Immunology: The Science of Self-NonselfDiscrimination (1982); Kennett, et al., Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses (1980); and Campbell,“Monoclonal Antibody Technology,” in Laboratory Techniques inBiochemistry and Molecular Biology (1984)).

“Antibody,” as used herein, is meant to include intact molecules as wellas fragments which retain their ability to bind to antigens (e.g., Faband F(ab′)₂ fragments). These fragments are typically produced byproteolytically cleaving intact antibodies using enzymes such as papain(to produce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). Theterm “antibody” also refers to both monoclonal and polyclonalantibodies. Polyclonal antibodies are derived from the sera of animalsimmunized with the antigen. Monoclonal antibodies can be prepared usinghybridoma technology (Kohler, et al., Nature 256:495 (1975); Hammerling,et al. in Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981)). In general, this technology involves immunizing ananimal, usually a mouse, with either intact TRHR-2 or a fragment derivedfrom TRHR-2. The splenocytes of the immunized animals are extracted andfused with suitable myeloma cells, e.g., SP₂O cells. After fusion, theresulting hybridoma cells are selectively maintained in HAT medium andthen cloned by limiting dilution (Wands, et al., Gastroenterology80:225-232 (1981)). The cells obtained through such selection are thenassayed to identify clones which secrete antibodies capable of bindingTRHR-2.

The antibodies, or fragments of antibodies, of the present invention maybe used to detect the presence of TRHR-2 protein using any of a varietyof immunoassays. For example, the antibodies may be used inradioimmunoassays or immunometric assays, also known as “two-site” or“sandwich” assays (see Chard, T., “An Introduction to Radioimmune Assayand Related Techniques,” in Laboratory Techniques in Biochemistry andMolecular Biology, North Holland Publishing Company, N.Y. (1978)). In atypical immunometric assay, a quantity of unlabelled antibody is boundto a solid support that is insoluble in the is fluid being tested, e.g.,blood, lymph, cellular extracts, etc. After the initial binding ofantigen to immobilized antibody, a quantity of detectably labelledsecond antibody (which may or may not be the same as the first) is addedto permit detection and/or quantitation of bound antigen (see e.g.Radioimmune Assay Method, Kirkham, et al., ed. pp. 199-206 (E&S.Livingstone, Edinburgh (1970)). Many variations of these types of assaysare known in the art and may be employed for the detection of TRHR-2.

Antibodies to TRHR-2 may also be used in the purification of either theintact receptor or fragments of the receptor (see generally, Dean, etal., Affinity Chromatography, A Practical Approach, IRL Press (1986)).Typically, antibody is immobilized on a chromatographic matrix such asSepharose 4B. The matrix is then placed in a column and the preparationcontaining TRHR-2 is passed through under conditions that promotebinding, e.g., under conditions of low salt. The column is then washedand bound TRHR-2 is eluted using a buffer that promotes dissociationfrom antibody, e.g., buffer having an altered pH or salt concentration.The eluted TRHR-2 may then be transferred into a buffer of choice, e.g.,by dialysis, and either stored or used directly.

IV. Assay for TRHR-2 Binding

One of the main uses for TRHR-2 nucleic acids and recombinant proteinsis in assays designed to identify agents, other than TRH, capable ofbinding to the TRHR-2 receptor. Such agents may either be agonists,mimicking the effects of TRH, or antagonists, inhibiting the effects ofTRH. Of particular interest is the identification of agents which bindto TRHR-2 receptors and increase the intracellular concentration ofcalcium in the cells. These agents have potential therapeuticapplication as analgesics; anesthetics; for reducing the damage due tospinal trauma; for controlling endocrine function; and for regulatinggastric secretion, particularly in the treatment of ulcers.

An example of an assay that may be used for detecting compounds bindingto TRHR-2 is presented in the examples and typical binding curves thatmay be obtained are shown in FIG. 4. The essential feature of the assaysis that a source of TRHR-2 is incubated together with a ligand known tobind to the receptor and with a compound being tested for bindingactivity. The preferred source for TRHR-2 is cells, preferably mammaliancells, transformed recombinantly to express the receptor. The cellsselected should not express a substantial amount of any other receptorwhich binds TRH, e.g., TRHR-1. This can easily be determined byperforming TRH binding assays on cells derived from the same tissue orcell lines as those recombinantly expressing TRHR-2 but which have notundergone transformation.

The assay may be performed either with intact cells or, alternatively,with membranes prepared from the cells (see e.g., Wang, et al., Proc.Natl. Acad Sci. USA 90:10230-10234 (1993)). The membranes are incubatedwith a ligand specific for TRHR-2 and with a preparation of the compoundbeing tested. After binding is complete, receptor is separated from thesolution containing ligand and test compound, e.g., by filtration, andthe amount of binding that has occurred is determined. Preferably, theligand used is TRH detectably labelled with a radioisotope. However,fluorescent or chemiluminescent labels can be used instead. Among themost commonly used fluorescent labelling compounds are fluorescein,isothiocynate, rhodamine, phycoerythrin, phycocycanin, allophycocyanin,o-phthaldehyde and fluorescamine. Useful chemiluminescent compoundsinclude luminol, isoluminol, theromatic acridinium ester, imidazole,acridinium salt, and oxalate ester. Any of these agents which can beused to detectably label TRH will produce a ligand suitable for use inthe assay.

Nonspecific binding may be determined by carrying out the bindingreaction in the presence of a large excess of unlabelled ligand. Forexample, labelled TRH may be incubated with receptor and test compoundin the presence of a thousandfold excess of unlabelled TRH. Nonspecificbinding should be subtracted from total binding, i.e., binding in theabsence of unlabelled TRH, to arrive at the specific binding for eachsample tested. Other steps such as washing, stirring, shaking, filteringand the like may be included in the assays as necessary. Typically, washsteps are included after the separation of membrane-bound ligand fromligand remaining in solution and prior to quantitation of the amount ofligand bound, e.g., by counting radioactive isotope. The specificbinding obtained in the presence of tests compound is compared with thatobtained in the presence of labelled ligand alone to determine theextent to which the test compound has displaced TRH.

In performing binding assays, care must be taken to avoid artifactswhich may make it appear that a test compound is interacting with theTRHR-2 receptor when, in fact, binding is being inhibited by some othermechanism. For example, the compound being tested should be in a bufferwhich does not itself substantially inhibit the binding of TRH to TRHR-2and should preferably be tested at several different concentrations.Preparations of test compound should also be examined for proteolyticactivity, and it is desirable that antiproteases be included in assays.Finally, it is desirable that compounds identified as displacing thebinding of ligand to TRHR-2 receptor be re-examined in a concentrationrange sufficient to perform a Scathard analysis on the results. Thistype of analysis is well known in the art and can be used fordetermining the affinity of a test compound for receptor (see e.g.,Ausubel, et al., Current Protocols in Molecular Biology, 11.2.1-11.2.19(1993); Laboratory Techniques in Biochemistry and Molecular Biology,Work, et al., ed., N.Y. (1978), etc.). Computer programs may be used tohelp in the analysis of results (see e.g., Manson, Methods Enzymol.92:543-577 (1983); McPherson, Kinetic, EBDA Ligand, Lowry-A Collectionof Radioligand Binding Analysis Programs, Elsevier-Biosoft, U.K.(1985)).

Assays for determining changes in second messenger, e.g. changes inintracellular calcium concentration, may be performed using compoundsthat have been identified as a result of their ability to bind toTRHR-2. These assays may be carried out as discussed in the examples orusing other methods for determining intracellular calcium concentration.Typically, calcium concentration assays will be performed separatelyfrom binding assays, but it may also be possible to perform binding andcalcium concentration assays on a single preparation of cells. TRHR-2binding compounds that stimulate an increase in intracellular calcium incells are agonists of TRH and should mimic its biological effects. Incontrast, compounds that specifically bind to TRHR-2 receptors but whichdo not increase intracellular calcium are antagonists of TRH and shouldinhibit its biological effects.

V. Assay for Ability to Modulate TRHR-2 Expression

One way to either increase or decrease the biological effects of TRH isto alter the extent to which TRHR-2 is expressed in cells. Therefore,assays for the identification of compounds that either inhibit orenhance expression of TRHR-2 are of considerable interest. These assaysare carried out by growing cells expressing TRHR-2 in the presence of atest compound and then comparing receptor expression in these cells withcells grown under essentially identical condition but in the absence ofthe test compound. As in the binding assays discussed above, it isdesirable that the cells used be substantially free of receptors for TRHother than TRHR-2. Scatchard analysis of binding assays performed withdetectably labelled TRH can be used to determine receptor number.

The binding assays may be carried out as discussed above in section IVand will preferably utilize cells that have been engineered torecombinantly express TRHR-2 as described in sections I and II. Ideally,the expression of TRHR-2 protein is controlled by the naturallyoccurring TRHR-2 regulatory element, e.g., the promoter which regulatescellular TRHR-2 expression in vivo.

A preferred group of test compounds for inclusion in the TRHR-2expression assay consists of oligonucleotides complimentary to varioussegments of the TRHR-2 nucleic acid sequence. These oligonucleotidesshould be at least 15 bases in length and should be derived fromnon-conserved regions of the receptor nucleic acid sequence.

Oligonucleotides which are found to reduce receptor expression may bederivatized or conjugated in order to increase their effectiveness. Forexample, nucleoside phosphoro-thioates may be substituted for theirnatural counterparts (see Cohen, Oligodeoxynucleotides, AntisenseInhibitors of Gene Expression, CRC Press (1989)). The oligonucleotidesmay be delivered to a patient in vivo for the purpose of inhibitingTRHR-2 expression. When this is done, it is preferred that theoligonucleotide be administered in a form that enhances its uptake bycells. For example, the oligonucleotide may be delivered by means ofliposome or conjugated to a peptide that is ingested by cells (see e.g.,U.S. Pat. Nos. 4,897,355 and 4,394,448; see also non-U.S. patentdocuments WO 89/03849 and EP0263740). Other methods for enhancing theefficiency of oligonucleotide delivery are well known in the art and arealso compatible with the present invention.

Having now described the invention, the same will be more readilyunderstood through reference to the following examples which areprovided by way of illustration and which are not intended to limit thescope of the invention.

EXAMPLES Example 1

Cloning and Sequencing of a Rat (TRHR-2) Thyrotropin-Releasing HormoneReceptor

A. Cloning and Sequencing Procedures

In order to isolate novel cDNA sequences encoding G protein-coupledreceptors, a PCR-based homology screening strategy was used. Rat spinalcord mRNA was isolated using the FastTrack™ kit (InVitrogen, San Diego,Calif.). Candidate sequences likely to encode G protein-coupledreceptors were amplified from this mRNA by reverse transcription PCRusing the following primers:

TM3-4: 5′-AT(C or T)(A or G)(C or G)(C or T)(A or G)TIGAI(A or C)G(A orG)TA-3′ (SEQ ID NO:3)

TM7-4: 5′-(A or C)(A or T)GG(C or T)(A or G)TAGAI(C or G)AI(A or C)GG(Aor G)TT-3′ (SEQ ID NO:4)

The templates for PCR amplification were synthesized using GeneAmp RNAPCR kits (N808-0017 Perkin Elmer) together with 200 ng of spinal cordpoly A⁺ RNA. One aliquot of the produced cDNA was then amplified with200 pmoles each of TM3-4 and TM7-4 primers and 2.5 units of Taq DNApolymerase in 50 mM KCl, 1.5 mM MgCl₂, 10 mM Tris(HCl), 200 μM dNTPs, pH9.0. The reaction tubes were heated at 95 degrees C. for one minute andsubjected to 39 cycles of denaturation (95 degrees C./min), annealing(42 degrees C./min) and extension (72 degrees C./min).

The amplified fragments were analyzed and size fractionated on a 1%agarose gel. Fragments between 500 bp and 800 bp were excised from thegel, purified using the Sephaglas BandPrep™ kit from Pharmacia (cat#27-9285-01), and inserted into the pGEM-T vector from Promega (cat#A3600). Recombinant pGEM-T clones were selected randomly and plasmid DNAwas prepared using the alkaline lysis method starting with 10 ml ofbacterial culture. The DNA sequence from these clones was determinedusing the Sanger dideoxynucleotide chain termination method on denatureddouble-stranded plasmid templates (Sanger et al., Proc. Natl. Acad. Sci.USA 74:5463-5437 (1977)), using the T7 sequencing kit from Pharmacia(cat# 27-1682-01). The clone pGemT-1-75 showed marked sequencehomologies with known GPCRs. The most homologous sequence was the humanand rat thyrotropin release hormone receptors (TRHR) (Matre et al.,Biochem. Biophys. Res. Comm. 195:179-185 (1993); Hinuma et al., Bioch.Biophys. Acta 1219:251-259 (1994); Duthie et al., Mol Cell. Endocrinol.95:R11-R15 (1993)), although a perfect match was not identified.

The insert DNA fragment of clone pGemT-1-75 was excised from the vectorusing Pst I and Sac II, isolated from an agarose gel and labeled with³²P by random primed synthesis using the Ready-To-Go™ DNA labeling kit(27-9251-01) from Pharmacia (Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory (2nd ed. 1989)). Thisprobe was used to screen a rat brain stem-spinal cord cDNA library in λZAP II (Stratagene, cat# 936521). The filters were incubated with theprobe for 18 hours at 65° C. in 2×SSC, 5×Denhardt's solutions and 0.2%SDS. The filters were rinsed twice in 0.1×SSC, 0.2% SDS at roomtemperature. They were then washed twice for 45 min in 0.1×SSC, 0.2% SDSat 65° C., once for 45 min at 65° C. in 5 mM EDTA, 0.2% SDS, pH 8.0 andfinally using 0.1×SSC at room temperature.

Hybridization-positive phages were purified and their inserts rescued byhelper phage mediated excision to yield plasmid DNA (Murray et al., Mol.Gen. Genet. 150:53-61 (1977); Schweinfest et al., Genet. Anal. Tech.Appl. 7:64-70 (1990)). The insert of plasmid pBS/TRHR2 was sequencedafter having generated a series of overlapping clones using theErase-A-Base kit from Promega (cat# 65740).

B. Results

An open reading frame of 352 amino acids was detected flanked by 3′ and5′ untranslated regions of, respectively, 183 and 361 bp. The sequenceof the open reading frame is displayed in FIG. 1. The relative molecularmass of the predicted protein is 39,500 daltons. Hydropathy analysis ofthe encoded protein is consistent with a topography of seventransmembrane domains indicative of the G protein-coupled receptorfamily (Sprengel et al., “Hormone Receptors,” in Handbook of Receptorsand Channels: G Protein-Coupled Receptors, pp. 153-207, CRC Press(1994)). In addition, sequence analysis revealed that the open readingframe of clone pBS-TRHR2 contains several conserved structural featuresfound among the members of the neuropeptide receptor family, including:an asparagine in TM1 (Asn40); a leucine (Leu64) and an aspartic acid(Asp 68) in TM2; and a serine (Ser109), an arginine (Arg120) and atyrosine residue (Tyr121) in TM3. Other features of this TRHR-2 receptorgene are the presence of a potential site for N-glycosylation in theamino terminus (Asn6) and the presence of several serines and threoninesin the carboxyl terminus and third intracellular loop, which may serveas potential sites for phosphorylation by protein kinases.

The overall sequence homology between TRHR-2 and the known rat TRHreceptor is 50.6%. However the sequence homology is higher in theputative transmembrane domains. Respectively, the homologies between theknown rat TRH receptor (TRHR-1) and TRHR-2 in TM1 to TM7 are 61%, 80%,74%, 58%, 52%, 77% and 71%.

Example 2

Transient Transfection Experiments

To generate a mammalian expression vector, a 1.3 Kb StuI-Xbalrestriction fragment, from pBS/TRHR2 was isolated and subcloned betweenthe Xba I and Eco RV sites of pCDNA3 (InVitrogen, San Diego, Calif.).This expression vector was called pCDNA3-TRHR2. Plasmid DNA for furtheranalysis was prepared using the Qiaprep system from Qiagen.

HEK293s cells were obtained form the Cold Spring Harbor laboratory. Theywere inoculated in 6-well plates (4×10⁵ cells per well) or in 10 cmPetri dishes (2×10⁶ cells per dish) in Dulbeco's Modified EssentialMedium (DMEM, Gibco BRL, cat# 11995-032) supplemented with 10% fetalbovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25μg/ml fungizone. One day after inoculation, the cells were transientlytransfected using a modified CaCl₂ method. Three and a half mg ofplasmid DNA per well or 20 μg per 10 cm petri dish was used. The cellswere harvested 48 hours post transfection for ligand binding or signaltransduction experiments. When transfected into HEK293 cells,pCDNA3-TRHR2 generated the expression of specific ³H-TRH binding sites.No specific ³H-TRH binding sites were generated by the transfection ofthe vector itself or a control pCDNA3 expression construct encoding anopioid receptor.

Example 3

Radioligand Binding to Stably Transfected Cells

A. Binding Assays

The TRH binding assay was performed on whole cells. Transfected cellswere washed twice with HBSS (Gibco BRL, cat# 14065) supplemented with0.05% bovine serum albumin, detached by gentle pipetting and aliquotedin eppendorf tubes for binding assays. One twentieth of the cellscollected from a confluent 75 cm² flask was used per assay point. Thebinding reaction was performed in a total volume of 300 ml of bindingbuffer (HBSS+0.05% bovine serum albumin) containing the transfectedcells and 10 nM of³H-thyrotropin releasing hormone (PeninsulaLaboratories Inc, cat # 7501) with or without unlabelled competitors.Non-specific binding was estimated in the presence of 1 mM of unlabelledTRH. Reactions were carried out for 60 min at room temperature andreactions were stopped by filtration through Unifilters-96 GF/B filters(Canberra Packard cat # 6005177) using the 96-well Filtermat 196filtration system from Canberra Packard. The filters were washed 3 timeswith 1 ml of washing buffer (50 mM Tris(HCl), 3 mM MgCl₂, pH 7.0). Theywere then dried at 55° C. for one hour and 50 μl of mScint-20 (CanberraPackard) was added per well. Filters were counted with the Topcountmicroplate counter from Canberra Packard.

B. Production of Stably Transfected Cells

HEK293s cells in a 10 cm petri dish were transfected with 20 mgpCDNA3-TRHR2. After 14 days of selection in culture medium containing600 μg/ml G418, resistant colonies were pooled. Then, single clones werepurified by 2 rounds of limited dilution in 96 well plates. Clones ofHEK293s cells expressing different levels of TRHR-2 receptor wereselected using a 3H-TRH binding assay.

C. Results

A binding reaction was performed using HEK 293 cells expressing TRHR-2.A single class of saturable ³H-TRH binding site was detected displayingan estimated Kd for ³H-TRH of 2.5 nM and a B_(max) of 430 fmol/mgproteins. Various TRH-related peptides were used in competitionexperiments. These experiments were performed using ³H-TRH as a tracerand revealed IC₅₀ values of 2.2 nM for pGlu-3 methyl-His-Pro-amide, 8.3nM for TRH, 640 nM for pGlu-His-Pro-Gly, and >10000 nM forpGlu-Glu-Pro-amide (Table 1).

TABLE 1 Binding Parameters of TRH Receptors* 293S/TRH-R clonal cell lineGH4Cl Cells B_(MAX) (fmol/mg) 534.56 ± 70.82 577.48 ± 80.77 Kd (nM):³H-TRH 6.17 ± 1.51 5.32 ± 1.63 IC₅₀ (nM): TRH (pGlu-His-Pro-amide) 8.39± 2.31 5.33 ± 2.81 IC₅₀ (nM): pGlu-Glu-Pro-amide >>10000 >>10000 IC₅₀(nM): pGlu-His-Pro-Gly 640.67 ± 75.19 >>10000 IC₅₀ (nM): 2.20 ± 0.710.43 ± 0.27 pGlu-3-methyl-His-Pro-amide *Three independent experimentswere performed, each with duplicate data points.

Example 4

Functional Assay: Intracellular Calcium Mobilization

A. Assay Procedure

Intracellular calcium concentration changes where determined byfluorescence measurement of the intracellular, calcium-sensitiveindicator Fura 2. Briefly, HEK293s cells were grown on glass cover slipsin culture medium at 37° C., 5% CO₂ and diluted 10 fold every 3 days.The cells were loaded at room temperature for 30 min using 2 mM of isFura 2/AM in simplified Grace's solution (SGS buffer) physiologicalbuffer (NBS: 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl₂, 1.2 mM MgSO₄, 10 mMHEPES, pH 7.4). Fura 2/AM is a membrane permeant, calcium-insensitiveester of Fura 2. After three washes in Fura-2 /AM-free buffer, the cellswere incubated for 15 to 30 min in SGNBS at room temperature to insurefull hydrolysis of the Fura 2 ester. The calcium-sensitive, hydrolyzedform of Fura 2 remains trapped intracellularly. Experiments wereconducted at room temperature on single cells or small groups of 3 to 7cells in a coverslip holder fitted to the stage of an IMT-2 invertedmicroscope equipped with a 40×-epifluorescence objective (UVFL40, 0.85,Olympus Optical Co, Tokyo) and coupled to a PTI ratio fluorescencesystem (Photon Technology International, London, Ontario, Canada).Sample illumination was provided by a 75 W xenon light source attachedto a filter-based light chopper unit which provided 340 and 380 nmexcitation wavelength alternating at a frequency of 5 Hz. The emittedlight was passed through an adjustable rectangular aperture followed bya 505 nm interference filter (10-nm bandwidth), and its intensity wasrecorded by a photon counter detector. Dye leakage, as determined byloss of fluorescence over a period of 30 min, was undetectable at bothexcitation wavelengths.

The ratio of the fluorescence intensity at the two excitationwavelengths can be converted into an estimate of ionized intracellularcalcium concentration by use of the formula:[Ca²⁺]_(i)=Kd×(Fmin/Fmax)×(R-Rmin)/(Rmax-R), where R, Rmin, and Rmax arethe fluorescence ratios recorded during the experiment (R) and duringcalibration tests on unlysed cells using 4 mM ionomycin in SGNBS (Rmax),followed by 10 mM EGTA addition at pH 8.2 (Rmin). Fmin and Fmax are thecorresponding fluorescence intensities for the 380 nm excitation and Kdis the Fura-2 dissociation constant at room temperature (135 nM).

Small volumes (10-50 μl) of drugs (hormones, agonists,neurotransmitters, TRH, bradykinin) from prediluted stock solutionsprepared in the appropriate buffer solutions were directly added to theexperimental chamber.

B. Results

Exposure of HEK293/TRHR-2 cells, but not wild type HEK 293 cells, tonanomolar concentrations of TRH resulted in a marked transient increasein the intracellular calcium concentration. The peak of [Ca⁺⁺]_(i)concentration was reached after about 1 second and the baseline[Ca⁺⁺]_(i) was attained after about 1 minute. TRH did not require thepresence of extracellular calcium to evoke the transient raise in[Ca⁺⁺]_(i).

Example 5

Northern Blot Analysis

A rat Multiple Tissue Northern blot (Clonetech (cat# 7764-1) was used tostudy the distribution of TRHR-2 in various tissues. The blot contained2 mg of rat poly(A)⁺ mRNA isolated from heart, brain, spleen, lung,liver, skeletal muscle, kidney and testes. The blot was firstpre-hybridized at 42° C. for three hours in a solution containing 50%formamide, 5×SSPE, 10×Denhardt's solution, 100 μg/ml sheared anddenatured salmon sperm DNA and 2% SDS. A radiolabelled probe wasprepared using Ready-to-go DNA labelling kit (Pharmacia BiotechCat.#27-9251-01) and the full length cDNA of the TRHR-2. Hybridizationwas carried out at 42° C. for about 18 hours in the solution describedabove. After an overnight hybridization, the blot was rinsed 2 times in2×SSC, 0.05% SDS at room temperature and then washed 2 times for 15minutes at room temperature followed by 2 washes at 50° C. for 15minutes each and then 2 washes at 60° C. in the same solution. The blotwas then exposed at −80° C. for 7 days to Kodak Biomax film withintensifying screens.

Expression of TRHR-2 mRNA was detected only in brain tissue. Theapparent size of the mRNA is about 8 kb kilobases. Other tissuescontained either no message or, at least, an insufficient amount ofmessage to be detected after one week exposure under the conditionsdescribed.

Example 6

In Situ Hybridization

A. Hybridization Procedure

Animals and Tissue Preparation

Adult male Sprague-Dawley rats (˜300 gm; Charles River, St-Constant,Quebec) were sacrificed by decapitation. Brain, pituitary and spinalcord were promptly removed, snap-frozen in isopentane at −40° C. for 20seconds and stored at −80° C. Frozen tissue was sectioned at 14 mm in aMicrom HM 500 M cryostat (Germany) and thaw-mounted onto ProbeOn Plusslides (Fisher Scientific, Montreal, Quebec). Sections were stored at−80° C. prior to in situ hybridization.

Riboprobe Synthesis

The plasmid pCDNA3-TRHR2 was linearized using either XbaI or HindIIIrestriction enzymes, which cut in the polylinker on either side of theinserted cDNA. Sense and antisense TRHR-2 riboprobes were transcribed invitro using either T7 or SP6 RNA polymerases (Pharmacia, Baie d'Urfe,Quebec), respectively in the presence of [³⁵S]UTP (˜800 Ci/mmol;Amersham, Oakville, Ontario). Following transcription, the DNA templatewas digested with DNAse I (Pharmacia). Riboprobes were subsequentlypurified by phenol/chloroform/isoamyl alcohol extraction andprecipitated in 70% ethanol containing ammonium acetate and tRNA. Thequality of labeled riboprobes was verified by polyacrylamide-urea gelelectrophoresis.

In Situ Hybridization

Sections were postfixed in 4% paraformaldehyde (BDH, Poole, England) in0.1 M phosphate buffer (pH 7.4) for 10 min at room temperature (RT) andrinsed in 3 changes of 2×standard sodium citrate buffer (SSC: 0.15 MNaCl. 0.015 M sodium citrate, pH 7.0).

Sections were then equilibrated in 0.1 M triethanolamine, treated with0.25% acetic anhydride in triethanolamine, rinsed in 2×SSC anddehydrated in an ethanol series (50-100%). Hybridization was performedin a buffer containing 75% formamide (Sigma, St. Louis, Mo.), 600 mMNaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 1×Denhardt's solution (Sigma), 50μg/ml denatured salmon sperm DNA (Sigma), 50 μg/ml yeast tRNA (Sigma),10% dextran sulfate (Sigma), 10 mM dithiothreitol and [³⁵S]UTP-labeledcRNA probes (10×10⁶ cpm/ml) at 55° C. for 18 h in humidified chambers.Following hybridization, slides were rinsed in 2×SSC at RT, treated with20 μg/ml RNase IA (Pharmacia) in RNase buffer (10 mM Tris, 500 mM NaCl,1 mM EDTA, pH 7.5) for 45 min at RT and washed to a final stringency of0.1×SSC at 65° C. Sections were then dehydrated and exposed to Biomax MRKodak film for 10 days. Neuroanatomical structures were identifiedaccording to the Paxinos and Watson rat brain atlas (Paxinos et al., TheRat Brain in Stereotaxic Coordinates, Academic Press (1986)).

B. Results

The most prominent labeling for TRHR-2 was detected throughout thethalamus, with the anterior, centromedian, centrolateral, paracentraland ventroposteromedian (VPM) nuclei exhibiting the highest intensity(see Table 2). In the more caudal thalamus, the medial geniculatenucleus was also moderately labeled. In addition, layers III-V of thecerebral cortex were moderately labeled. The pontine nuclei as well asthe Purkinje cell layer of the cerebellum also displayed a high densityof TRHR-2 mRNA hybridization. More moderate labeling was detected in themedial amygdalar nucleus as well as in a specific portion of the lateralhypothalamic area which does correspond to well known nuclearboundaries. Moderate to weak hybridization was detected throughout thereticular formation of the brain stem. Other cephalic areas such as thehippocampus, the remaining hypothalamus, the pituitary gland and basalganglia were generally devoid of labeling. In the spinal cord, TRHR-2mRNA expression was restricted to the entire dorsal horn. This is instark contrast to that of the known TRH receptor mRNA under the sameconditions which appears to be present only in the hypothalamus, theanterior pituitary gland and some sparse neurons in the ventral horn ofthe spinal cord.

TABLE 2 Localization of TRHR-1 and TRHR-2 by In Situ HybridizationTissue TRHR-1 TRHR-2 Pituitary Gland anterior +++ − intermediate − −posterior − − Spinal cord dorsal horn − ++ ventral horn + − Brainpiriform cortex + − amygdala + + hypothalamic nuclei + − thalamus − +++medial habenular n. − ++ frontal & parietal cx. − ++ pontine nuclei −+++ cerebellum − +++

5 1 1600 DNA Rat 1 ctttaaacca cagcctctca aatacgcatc cctacactggctcctttctt ggtcttccta 60 tctgagccct gatggcttct ccagctgctc ttccagagacctgggttcaa ttcccagcac 120 ctatatgaca acttacagat tggtggctgt aactccaatccgggggatgc aatgccatct 180 tctggcctcc agaggcacta catacacaca tgatacacagaatatacaca cgtgtatatt 240 taggtaaagt gcctgtgcac ataaaaaaaa ataaaaaggaaaaaaattaa atcagaagga 300 acaggcaccg gtcacttacc aaggtcaagg cctacagggcaccacagaaa acaccagcaa 360 gatggatggc cccagtaatg tctcgctcat tcacggtgacaccacgctgg gcctgccaga 420 gtacaaggtg gtctcagtct tcctagtgct cctggtgtgcaccctgggca tcgtgggcaa 480 tgccatggtg attctggtgg tgctgacctc acgtgacatgcacacaccca ccaactgcta 540 cctggtcagc ctggccctcg ctgacctcct cgtgctgctggctgcgggtc tgcccaatgt 600 ctctgacagc ctagtggggc actggatcta tggacgtgctggctgcttgg gcatcaccta 660 cttccagtac ctgggcatca atgtctcctc cttctccatcctggccttca ctgtggagag 720 gtatatagcc atttgccacc cactgagagc acagaccgtgtgcactgtgg cccgggccaa 780 acggatcatg gcaggcatct ggggggtcac gtccctctattgcctactct ggttcttcct 840 ggtggatctc aatgtccgtg acaaccagcg ccttgaatgtggctacaaag tgccccgagg 900 actctacctg cccatctacc tgctggactt cgctgtctttttcatcggac ccttgctggt 960 gaccctcgtg ctctatgggc tcatcgggag gattttatttcagagcccgt tgtcccagga 1020 agcctggcag aaggagaggc agccccatgg gcagagcgaggctgcaccag gcaactgctc 1080 cagggccaag agctccagga agcaggccac caggatgctggccgtggttg tgttgctttt 1140 tgccgtgctg tggacccctt accgcacact ggtactgctcaactcctttg tggcccagcc 1200 tttcctggac ccctgggtcc tgctgttctg ccgcacctgtgtctacacca acagcgctgt 1260 caaccctgtc gtctacagcc tgatgtcaca gaagttccgggcggccttcc tgaaactgtg 1320 ctggtgcagg gcagctgggc cacagcggag ggcagcacgcgtcctcacca gtaactacag 1380 tgccgcccag gagacctcag aaggaactga gaagatgtagctgggctcca gtgaggtctc 1440 aggtcccacg gcagcaggtc ccctggcctg tcagcatgagccctacttca gtgtgctctg 1500 aggactcccg cctggcccct gaccccgctt aaggcttggttggcatttgg gaggcatcag 1560 gagaggggca ggcagctcct tgcttatggg tttccagagg1600 2 352 PRT Rat 2 Met Asp Gly Pro Ser Asn Val Ser Leu Ile His Gly AspThr Thr Leu 1 5 10 15 Gly Leu Pro Glu Tyr Lys Val Val Ser Val Phe LeuVal Leu Leu Val 20 25 30 Cys Thr Leu Gly Ile Val Gly Asn Ala Met Val IleLeu Val Val Leu 35 40 45 Thr Ser Arg Asp Met His Thr Pro Thr Asn Cys TyrLeu Val Ser Leu 50 55 60 Ala Leu Ala Asp Leu Leu Val Leu Leu Ala Ala GlyLeu Pro Asn Val 65 70 75 80 Ser Asp Ser Leu Val Gly His Trp Ile Tyr GlyArg Ala Gly Cys Leu 85 90 95 Gly Ile Thr Tyr Phe Gln Tyr Leu Gly Ile AsnVal Ser Ser Phe Ser 100 105 110 Ile Leu Ala Phe Thr Val Glu Arg Tyr IleAla Ile Cys His Pro Leu 115 120 125 Arg Ala Gln Thr Val Cys Thr Val AlaArg Ala Lys Arg Ile Met Ala 130 135 140 Gly Ile Trp Gly Val Thr Ser LeuTyr Cys Leu Leu Trp Phe Phe Leu 145 150 155 160 Val Asp Leu Asn Val ArgAsp Asn Gln Arg Leu Glu Cys Gly Tyr Lys 165 170 175 Val Pro Arg Gly LeuTyr Leu Pro Ile Tyr Leu Leu Asp Phe Ala Val 180 185 190 Phe Phe Ile GlyPro Leu Leu Val Thr Leu Val Leu Tyr Gly Leu Ile 195 200 205 Gly Arg IleLeu Phe Gln Ser Pro Leu Ser Gln Glu Ala Trp Gln Lys 210 215 220 Glu ArgGln Pro His Gly Gln Ser Glu Ala Ala Pro Gly Asn Cys Ser 225 230 235 240Arg Ala Lys Ser Ser Arg Lys Gln Ala Thr Arg Met Leu Ala Val Val 245 250255 Val Leu Leu Phe Ala Val Leu Trp Thr Pro Tyr Arg Thr Leu Val Leu 260265 270 Leu Asn Ser Phe Val Ala Gln Pro Phe Leu Asp Pro Trp Val Leu Leu275 280 285 Phe Cys Arg Thr Cys Val Tyr Thr Asn Ser Ala Val Asn Pro ValVal 290 295 300 Tyr Ser Leu Met Ser Gln Lys Phe Arg Ala Ala Phe Leu LysLeu Cys 305 310 315 320 Trp Cys Arg Ala Ala Gly Pro Gln Arg Arg Ala AlaArg Val Leu Thr 325 330 335 Ser Asn Tyr Ser Ala Ala Gln Glu Thr Ser GluGly Thr Glu Lys Met 340 345 350 3 17 DNA Artificial Sequencemodified_base (9, 12) i 3 atyrsyrtng anmgrta 17 4 20 DNA ArtificialSequence modified_base (11, 14) i 4 mwggyrtaga nsanmggrtt 20 5 4 PRTArtificial Sequence MOD_RES (1) PYRROLIDONE CARBOXYLIC ACID 5 Glu HisPro Gly 1

What is claimed is:
 1. A substantially pure protein comprising the aminoacid sequence of SEQ ID NO:2, wherein said protein comprises a receptorfor thyrotropin-releasing hormone (TRH).
 2. A substantially pure proteinof claim 1, wherein said amino acid sequence is 352 residues in lengthand preferentially binds TRH.
 3. A substantially pure protein whereinsaid protein has an amino acid sequence that consists essentially of theamino acid sequence of SEQ ID No:2.
 4. A substantially pure protein ofclaim 3, wherein said protein preferentially binds TRH and is 352 aminoacids in length.
 5. A substantially pure protein of claim 2, whereinsaid protein has a sequence consisting of the amino acid sequence of SEQID NO:2.
 6. An isolated recombinant thyrotropin-releasing hormonereceptor, wherein said receptor is produced by transfecting a host cellwith an expression vector comprising a polynucleotide encoding theprotein of any one of claims 1-5 and expressing and isolating theprotein, and wherein the protein is the receptor.
 7. The recombinantthyrotropin-releasing hormone receptor of claim 6, wherein said hostcell is a eukaryotic cell.
 8. The recombinant thyrotropin-releasinghormone receptor of claim 7, wherein said eukaryotic cell is a mammaliancell.